2-methyl-p-phenylenediamine sulfate

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 10, 2011 to June 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

no

Remarks:

study was conducted in a lab that is in the process of attaining GLP status, and good scientific practices were followed

Test material

Test material information

Constituent 1

Reference substance name:

2-methyl-p-phenylenediamine sulfate

EC Number:

210-431-8

EC Name:

2-methyl-p-phenylenediamine sulfate

Cas Number:

615-50-9

Molecular formula:

C7H10N2.H2O4S

IUPAC Name:

2-methylbenzene-1,4-diamine; sulfuric acid

Constituent 2

Reference substance name:

p-toluenediamine sulfate

IUPAC Name:

p-toluenediamine sulfate

Details on test material:

The test substance of interest in this study was 2,5-diaminotoluene sulfate (PTD). The test substance was evaluated under realistic preparation and use in a hair dye formulation. As a result, the test materials in this study were mixtures of the test substance (2,5-Diaminotoluene sulfate; PTD) with 3 couplers, with or without H2O2, and with other key ingredients to reflect realistic and unique use conditions of oxidative dyes by the consumer, as well as releases from formulation plant conditions. The hair dye formulation tested was the darkest shade possible (it contained the highest amount of PTD). Details on preparation of coloration products are provided under 'Confidential details on test material' section. A table outlining the details of test material is provided under ‘Any other information on materials and methods incl. tables’ section below.
The details on test substance of interest (PTD) are as follows:
- Name of test material: 2, 5-Diaminotoluene sulfate (PTD; Colipa A005)
- TSIN: GTS96959
- Substance type: Pure active substance
- Physical state: Off white light pink
- Stability under test conditions: Not reported
- Storage condition of test material: Not reported

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TESTING SOLUTIONS
- Method: Six independent tests with three different coloration mixtures were conducted using (i) fresh coloration product, and (ii) aged coloration product.
1. 1000 mL of test solutions were prepared by dilution of stock solution (PTD-containing formulation) with blended water in 1000 mL volumetric flasks. The solution was allowed to stir for 10 min prior to aliquoting to individual test vessels. This solution was tested fresh.
2. Remaining solutions were allowed to stir overnight in the dark for 24 h at room temperature (to mimic the 24 hr time spent in sewer before reaching the sewage treatment plant) upon which tests were repeated with 24 h old solutions.
3. These 24 h aged solutions were again centrifuged for 15 min at 10000 g and supernatant was collected and used for preparing test solutions.
- Dilution water: ESD Blended water (South West well water diluted approx. 50:50 with reverse osmosis water)
- Control: Prepared using dilution water without test material

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Order: Cladocera
- Strain: Not reported
- Source: ARO, Aquatic Research Organisms, Hampton, New Hampshire.
- Age at study initiation: First instar Daphnia magna (neonates <24 hours old)
- Culture maintenance: Daphnids were kept in continuous batch culture using ESD blended water and were thinned to approx. 20 adults weekly. Daphnids were fed an algae mixture of batch cultured Ankistrodesmus falcactus, Chlamydomonas rheinhardtii, and Scenedesmus quadricauda. Daphnids utilized in tests were obtained from productive females held in groups in 2 L crystallizing dishes filled with approximately 1.5-1.75 L of the dilution medium. The day prior to testing, females were removed from batch cultures to isolate released young.
- Conditions of culture:
1. Temperature: 20.9˚C
2. Dissolved oxygen : 8.0 mg/L
3. pH: 8.19
4. Total water hardness: 181 mg/L
5. Total alkalinity: 142 mg/L

ACCLIMATION
- Acclimation period: Test conditions were the same as culture conditions.
- Acclimation conditions: Same as test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

None

Test conditions

Hardness:

Overall range: 170-210 mg/L as CaCO3.
Freshly made coloration product: 170 - 210 mg/L
24 h aged coloration product: 179 - 211 mg/L

Test temperature:

22 °C. The tests were conducted in a constant temperature room at 22 °C. All day 1 and day 2 temperature measurements were 22 °C.
Test solutions, as made, were typically >22 °C (range, 20.9 – 26.4 C), probably because of the temperature of the diluent, and the possible exothermic behavior of the hair dye mixtures. Test solutions quickly equilibrated to 22 °C once placed in constant temperature room.

pH:

Overall range: 7.6-8.5
Freshly made coloration product: 7.76 - 8.46
24 h aged coloration product: 7.61 - 8.49

Dissolved oxygen:

Overall range: 6.4-8.1
Freshly made coloration product: 6.6 - 8.1 mg/L
24 h aged coloration product: 6.4 - 8.1 mg/L

Nominal and measured concentrations:

Nominal test concentrations were:
(i) Coloration product containing PTD + couplers: 0, 0.05, 0.16, 0.49, 1.48, 4.44, 13.33 and 40 mg/L
(ii) Coloration product containing PTD + couplers + H2O2: 0, 0.025, 0.08, 0.25, 0.74, 2.22, 6.67 and 20 mg/L
(iii) Coloration product containing PTD + couplers + H2O2 + Hair: 0, 0.025, 0.08, 0.25, 0.74, 2.22, 6.67 and 20 mg/L
No details on measured concentrations are provided in the study report. The results are based on nominal test concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: Beakers
- Type: Open, loosely covered (with saran wrap) to reduce the loss of water due to evaporation and to avoid the entry of dust into the solutions
- Material, size, headspace, fill volume: 250 mL Glass beaker containing 100 mL test medium
- Aeration: No
- No. of organisms per vessel: 10
- No. of vessels per concentration: 2
- No. of vessels per control: 2
- Biomass loading rate: 1 daphnia/10 mL

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was ESD blended water, prepared by blending Southwest well water and reverse osmosis water together.
- Alkalinity: overall range 142-240 mg/L. Details on alkalinity are as follows:
Freshly made coloration product
(i) Coloration product containing PTD: 142 mg/L (0 h in control); 158 mg/L (48 h in Control); Alkalinity range in test vessels: 151- 240 mg/L
(ii) Coloration product containing PTD + H2O2: 142 mg/L (0 h in control); 155 mg/L (48 h in Control); Alkalinity range in test vessels: 148 - 212 mg/L
(iii) Coloration product containing PTD + H2O2 + Hair: 142 mg/L (0 h in control); 159 mg/L (48 h in Control); Alkalinity range in test vessels: 171- 181 mg/L
24 h aged coloration product
(i) Coloration product containing PTD: 150 mg/L (0 h in control); 154 mg/L (48 h in Control); Alkalinity range in test vessels: 142- 166 mg/L
(ii) Coloration product containing PTD + H2O2: 150 mg/L (0 h in control); 154 mg/L (48 h in Control); Alkalinity range in test vessels: 143 - 163 mg/L
(iii) Coloration product containing PTD + H2O2 + Hair: 150 mg/L (0 h in control); 154 mg/L (48 h in Control); Alkalinity range in test vessels: 142 - 171 mg/L
- Culture medium different from test medium: No
- Intervals of water quality measurement: Water quality parameters measured include dissolved oxygen, temperature, pH, hardness and alkalinity. Measurements for pH and temperature were taken from all concentrations, in addition to control, at 0 and 48 h, and from concentrations that had 100% mortality at 24 h. Temperature readings were taken directly from digital readouts on the incubator at the time of observation.
Measurements for dissolved oxygen, hardness and alkalinity were taken from the high and low concentrations, in addition to control, at 0 and 48 h as well as concentrations that had 100% mortality at 24 h.

OTHER TEST CONDITIONS
- Adjustment of pH: Yes, pH was adjusted in the range of 7.80 to 8.20 in freshly made and 24 h aged PTD containing product and in 24 h aged PTD + H2O2 + Hair containing product.
- Photoperiod: 16/8 light/dark cycle

EFFECT PARAMETERS MEASURED: Mortality or complete immobility of daphnids was determined in all groups at 24 and 48 h of exposure. A daphnid was considered to be immobile if it was not able to swim within 15 sec after gentle agitation of the test vessel.

TEST CONCENTRATIONS
- Dilution factor for preparation of test concentrations: 3
- Range finding study: Not reported

Reference substance (positive control):

yes

Remarks:

H2O2 (6% solution diluted 1:1 in blended water)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.19 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 1.05 - 1.34 mg/L (Freshly made coloration product containing PTD)

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.72 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 1.50 - 1.98 mg/L (Freshly made coloration product containing PTD + H2O2)

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.91 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 2.19 – 3.87 mg/L (Freshly made coloration product containing PTD + H2O2 + hair)

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.82 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 0.68 – 0.98 mg/L (aged coloration product containing PTD )

Effect concentrations 5

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.57 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 2.24 – 2.95 mg/L (aged coloration product containing PTD + H2O2)

Effect concentrations 6

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

6.77 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 6.11 – 7.50 mg/L (aged coloration product containing PTD + H2O2 + hair)

Details on results:

- Biological observations: For details on % mortality, refer to 'Table 2, 3 and 4,' under 'Any other information on results incl. tables' section.
- Mortality of control: 0%, with the exception that 5% mortality in the control for the freshly prepared coloration product containing PTD + H2O2 + hair.
- PTD, tested in the PTD + H2O2 + hair mixture, was the least toxic, while PTD, tested in the coloration product alone (no H2O2, no hair), was the most toxic. The lower toxicity of PTD + H2O2 + hair was explained by the fact that during the dyeing process a certain amount of coloration product deposits onto the hair.
- The 24 h aged coloration product containing PTD + H2O2 + hair exhibited lower toxicity than fresh coloration product containing PTD + H2O2 + hair.

Results with reference substance (positive control):

- Results with reference substance valid? Yes, results from the H2O2 control (1:1000 dilution) showed 100% mortality at 24 h.
- Results from the ammonium chloride control study generated a 48 h EC50 of 20.6 (14.7 - 44.0) mg/L and corresponded to data in the literature.

Reported statistics and error estimates:

- Data was analyzed by appropriate statistical methods to calculate an EC50 with 95% confidence limits.
- Probit analysis was the preferred method followed by the non-linear interpolation method of Bruce and Versteeg (1992).
- These toxicity tests provided suitable data for trend analysis, but do not allow comparisons with respect to statistical significance. Overlapping confidence limits among the various tests may suggest similarity, non-overlapping confidence limits may suggest differences, but these will not be construed as tests of statistical significance.

Any other information on results incl. tables

Results on ammonia toxicity study

- Measured ammonia concentrations trended toward expected values for an open system. Lower concentrations approached the level of detection of the method and are not included in this report but results are as expected.

- Ammonia concentrations at 24 h were significantly lower conceivably due to a number of reasons such as evaporation, biological uptake, chemical interactions, etc. Ammonia concentrations can contribute to the toxicity profile of the tested coloration product, which means that the toxicity of PTD, as measured in this study, may be over-estimated.

Table 1: Summary on water quality (Study # 78084)

Test Material	Water quality
Identity	pH			Hardness (mg/L)		Temperature (˚C)		Dissolved Oxygen (mg/L)		Alkalinity (mg/L)
	Control		Test	Control	Test	Control	Test	Control	Test	Control	Test
Freshly prepared coloration product
CP containing PTD + couplers		8.19 (at 0 h)       8.43 (at 48 h)	7.90 – 8.41	181 (at 0 h)       206 (at 48 h)	170 -210	20.9 (at 0 h)       22.0 (at 48 h)	22.0 – 25.9	8.0 (at 0 h)       8.0 (at 48 h)	6.6 – 8.0	142 (at 0 h)     158 (at 48 h)	151- 240
CP containing PTD + couplers + H2O2			7.76 – 8.46		183 -205		22.0 – 26.4		6.7 – 8.0		148 - 212
CP containing PTD + couplers + H2O2 + Hair			7.84 – 8.43		189 -205		22.0 – 26.0		6.7 – 8.1		171- 181
24 h aged coloration product
CP containing PTD + couplers		7.61 (at 0 h)       8.49 (at 48 h)	7.77 – 8.49	191 (at 0 h)       202 (at 48 h)	179 -211	23.4 (at 0 h)       22.0 (at 48 h)	22.0 – 25.5	6.4 (at 0 h)       8.0 (at 48 h)	7.1 – 8.0	150 (at 0 h)     154 (at 48 h)	142 - 166
CP containing PTD + couplers + H2O2			7.76 – 8.49		194 -210		22.0 – 25.8		7.0 – 8.1		143 - 163
CP containing PTD + couplers + H2O2 + Hair			7.77 – 8.48		192 -212		22.0 – 26.1		6.8 – 8.0		142 - 171

 

Table 2: Effect of Coloration product containing PTD + couplers to Daphnia magna: Acute toxicity (study # 78084)

Dilution (1:x)	PTD concentration (mg/L)	Number of organisms at 0 h	24 h % mortality	48 h % mortality
Freshly prepared coloration product
0	0	20	0	0
1000	40	20	100	100
3000	13.33	20	100	100
9000	4.44	20	55	100
27000	1.48	20	20	65
81000	0.49	20	0	15
243000	0.16	20	0	5
729000	0.05	20	0	0
24 h aged coloration product
0	0	20	0	0
1000	40	20	100	100
3000	13.33	20	100	100
9000	4.44	20	20	100
27000	1.48	20	15	90
81000	0.49	20	0	15
243000	0.16	20	0	0
729000	0.05	20	0	5

 

Table 3: Effect of Coloration product containing PTD + couplers + H2O2 to Daphnia magna: Acute toxicity (study # 78084)

Dilution (1:x)	PTD concentration (mg/L)	Number of organisms at 0 h	24 h % mortality	48 h % mortality
Freshly prepared coloration product
0	0	20	0	0
1000	20	20	100	100
3000	6.67	20	100	100
9000	2.22	20	45	75
27000	0.74	20	0	10
81000	0.25	20	0	10
243000	0.08	20	0	5
729000	0.025	20	0	5
24 h aged coloration product
0	0	20	0	0
1000	20	20	100	100
3000	6.67	20	45	100
9000	2.22	20	0	40
27000	0.74	20	0	15
81000	0.25	20	0	0
243000	0.08	20	0	5
729000	0.025	20	0	0

 

Table 4: Effect of Coloration product containing PTD + couplers + H2O2 + Hair to Daphnia magna: Acute toxicity (study # 78084)

Dilution (1:x)	PTD concentration (mg/L)	Number of organisms at 0 h	24 h % mortality	48 h % mortality
Freshly prepared coloration product
0	0	20	5	5
1000	20	20	100	100
3000	6.67	20	50	95
9000	2.22	20	0	0
27000	0.74	20	0	0
81000	0.25	20	0	10
243000	0.08	20	5	20
729000	0.025	20	0	0
24 h aged coloration product
0	0	20	0	0
1000	20	20	65	100
3000	6.67	20	5	50
9000	2.22	20	0	10
27000	0.74	20	0	5
81000	0.25	20	0	5
243000	0.08	20	0	0
729000	0.025	20	0	0

All results are based on nominal concentrations.

PTD = 2,5-diaminotoluene sulfate (CAS # 615-50-9)

Couplers = resorcinol (CAS # 108-46-3), m-aminophenol (CAS # 591-27-5), 4-amino-2-hydroxytoluene (CAS # 2835-95-2).

H2O2 = hydrogen peroxide

 

Table 5: Results with Ammonium chloride (study # 78084)

Dilution (1 :X)	Concentration (mg/L)	Number of organisms at 0 h	24 h % mortality	48 h % mortality
Ammonia 1000	44.0	20	100	100
3000	14.67	20	0	20
9000	4.89	20	0	0
27000	1.63	20	0	0
H2O2 1000	-	20	100	100

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 48 h EC50 of 2,5-diaminotoluene sulfate (PTD) to Daphnia magna, tested in hair coloration formulations, freshly-made and 24 hr old, was as follows, based on nominal concentration:
(i) Coloration product containing PTD: 0.82 - 1.19 mg/L.
(ii) Coloration product containing PTD + H2O2: 1.72 -2.57 mg/L.
(iii) Coloration product containing PTD + H2O2 + hair: 2.91 – 6.77 mg/L.

Executive summary:

The 48 h acute toxicity of 2,5-diaminotoluene sulfate (PTD) to Daphnia magna, tested in hair coloration formulations, was determined under static conditions, following the OECD 202 guideline. The purpose of this study was to determine the toxicity of PTD (the test substance of interest) in a representative hair dye formulation to Daphnia magna under (a) consumer use conditions, and (b) release conditions at the formulation plant. Therefore, the test solutions evaluated in this study were mixtures of PTD with couplers, with or without H2O2, and with or without exposure to hair. The details of the test design are as follows:

(i) Coloration product containing PTD + couplers (mixture mimicking the effluent of a formulation plant)

(ii) Coloration product containing PTD + couplers + H2O2 (mixture mimicking preparation by consumer)

(iii) Coloration product containing PTD + couplers + H2O2 + Hair (mixture mimicking actual dyeing process at consumer level).

Six independent tests with three different coloration mixtures were conducted using (i) fresh coloration product and (ii) 24 h aged coloration product
.There were two replicates at each test concentration and control. The nominal test concentrations of test material (PTD) were as follows:

(i) Coloration product containing PTD + couplers: 0, 0.05, 0.16, 0.49, 1.48, 4.44, 13.33 and 40 mg/L

(ii) Coloration product containing PTD + couplers + H2O2: 0, 0.025, 0.08, 0.25, 0.74, 2.22, 6.67 and 20 mg/L

(iii) Coloration product containing PTD + couplers + H2O2 + Hair: 0, 0.025, 0.08, 0.25, 0.74, 2.22, 6.67 and 20 mg/L.

Results are based on nominal test concentrations. Endpoints evaluated were mortality or complete immobility after 48 h of exposure.

The 48 h EC50, based on nominal concentrations, were as follows:

(i) Coloration product containing PTD: 1.19 mg/L (freshly made); 0.82 mg/L (24 h old)

(ii) Coloration product containing PTD + H2O2: 1.72 mg/L (freshly made); 2.57 mg/L (24 h old)

(iii) Coloration product containing PTD + H2O2 + hair: 2.91 mg/L (freshly made); 6.77 mg/L (24 h old).

Results with the PTD-containing coloration products, and those from literature ecotoxicity data on PTD itself, showed that these very processes (contact with other ingredients and contact to hair) significantly lowered the acute toxicity of PTD compared to that observed in laboratory tests of PTD itself. Tested alone as a single substance, the acute toxicity of PTD was 0.74 mg/L (literature value). Tested in a coloration product, the acute toxicity of PTD was 1.0 mg/L (average of fresh and 24 hr old). Tested in a coloration product + H2O2, mimicking preparation by the consumer, the acute toxicity of PTD was 2.2 mg/L (average of fresh and 24 hr old). Tested in a coloration product + H2O2 + exposure to hair, mimicking the hair dying process by the consumer, the acute toxicity of PTD was 4.8 mg/L (average of fresh and 24 hr old).

This acute toxicity test is classified as acceptable and satisfies the guideline requirement for the current OECD 202 (Daphnia sp. acute Immobilization test).